Transforming growth factor-alpha stimulates enterocyte proliferation and accelerates intestinal recovery following methotrexate-induced intestinal mucositis in a rat and a cell culture model.
OBJECTIVE
Recent evidence suggests that transforming growth factor-alpha (TGF-alpha) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-alpha on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells.


METHODS
Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-alpha. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 microg/kg) of MTX given intraperitoneally, and MTX-TGF-alpha rats treated with one dose of MTX followed by two doses of TGF-alpha (75 microg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park's score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal-Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant.


RESULTS
The in vitro experiment demonstrated that treatment with TGF-alpha of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-alpha resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-alpha rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-alpha rats corresponded with the increase in ERK protein levels in intestinal mucosa.


CONCLUSIONS
Treatment with TGF-alpha prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-alpha also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.
Sukhotnik
Igor
I
Laboratory of Intestinal Adaptation and Recovery, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. igor-dr@internet-zahav.net
Shteinberg
Dan
D
Ben Lulu
Shani
S
Bashenko
Yulia
Y
Mogilner
Jorge G
JG
Ure
Benno M
BM
Shaoul
Ron
R
Shamian
Benhoor
B
Coran
Arnold G
AG
D000818:Animals; D018938:Caco-2 Cells; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D020895:Enterocytes / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects; D008297:Male; D008727:Methotrexate; D052016:Mucositis / Q000139:chemically induced / Q000188:drug therapy*; D009676:Noxae; D051381:Rats; D017207:Rats, Sprague-Dawley; D020127:Recovery of Function; D016211:Transforming Growth Factor alpha / Q000494:pharmacology*
D036341:Intercellular Signaling Peptides and Proteins; D009676:Noxae; D016211:Transforming Growth Factor alpha; D008727:Methotrexate